GSM8047419: Riboseq_KO_water_Rep2; Homo sapiens; OTHER
Cells at 80-90% confluency were harvested from a 15 cm dish. Cells were incubated with 1 µM thapsigargin for 2hrs, and then treated with 50 µg/mL cyclohexamide (CHX) for 1 minute. Then, cells were washed gently with PBS containing 50 µg/mL CHX and lysed in ice-cold lysis buffer (20mM Tris pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1% v/v Triton x-100, 1mM DTT, 20-25 U/mL TURBO DNAse (Thermo Fisher Scientific, AM2238), 100 µg/mL CHX). Cells were triturated with a 26-gauge needle. Lysate was recovered after spinning for 10 min, 20,000g, at 4°C. Lysates were treated with RNAse I (Ambion, 100 U/µL) at room temperature for 45 minutes in slow agitation. Then, treated with SUPERase Inhibitor (Ambion, 20 U/µL) on ice. Monosomes were recovered by size exclusion chromatography (Illustra MicroSpin Columns, S-400 HR, VWR, 95017-619), and footprint RNA fragments were extracted from the flow-through using a Direct-zol kit (Zymo Research). Gel slices of RNA fragments with sizes between 26-34 nt were excised from a 15% polyacrylamide TBE-Urea gel (Thermo Fisher Scientific, EC68855BOX). Eluted RNA was treated with T4 PNK and preadenylated linker was ligated to the 3' end using T4 RNA ligase 2 truncated KQ (NEB, M0373L). Linker-ligated RNA was reverse transcribed with Protoscript II (NEB, M0368L) for 30 minutes at 50°C. Template RNA was hydrolyzed with 1M NaOH for 20 minutes at 70°C. Gel slices of reverse transcribed cDNA around 105 nt long, were excised from 15% polyacrylamide TBE-Urea gel. Eluted cDNA was circularized with CircLigase II ssDNA ligase (Lucigen, CL9021K) for 1 hr at 60°C. rRNA was depleted with biotinylated oligos18. cDNA libraries were amplified with different reverse indexing primers per sample.